Coagulation assays and reagents

ABSTRACT

Novel assays and reagents for determining coagulative properties of blood or plasma are disclosed, as are agents and methods for stemming bleeding. Novel ellagic acid-based activators consist essentially of aqueous solutions of ellagic acid, phenol and suitable metal ions and give defined values for a novel platelet assay also disclosed. Inventive coagulation reagents including propyl gallate or tannin are also disclosed, as are aPTT reagents having sensitivity to heparin and Factor deficiencies far superior to prior aPTT reagents. Disclosed heparin-sensitive reagents include dextran sulfate. Also disclosed is applicant&#39;s discovery that reagents including hydroxy-substituted compounds and metal ions, heretofore known to activate Hagemann Factor, also cause platelets to release Platelet Factor 3, and a series of platelet-sensitive tests found to be enabled by this discovery. These platelet-sensitive tests include assays for platelet activity, systemic lupus, platelet reserve, which can signify type II lipoprotein defects, and for Platelet Factor 4. Disclosed methods and materials for stemming bleeding center around applicant&#39;s discovery that bleeding from bleeding sites can be stopped up to six time faster by applying to the site a hemostatic agent including effective amounts of hydroxy-substituted aromatic compound and metal ion.

This application is a division of application Ser. No. 08/158,838, filedon Nov. 29, 1993 now U.S. Pat. No. 5,451,509, which is a division ofapplication Ser. No. 07/946,811, filed on Sep. 16, 1992 now U.S. Pat.No. 5,451,509, which is a continuation of Ser. No. 07/510,178 filed onApr. 17, 1990.

BACKGROUND OF THE INVENTIONS

The present inventions relate generally to medical diagnosis andtreatment, and more particularly to assays and reagents for determiningcoagulative properties of blood or plasma, and to agents and methods forpromoting clotting.

By way of background, the ability of animals to selectively form bloodclots in areas of trauma is a vital function. Failure of the blood toclot, of course, can lead to severe hemorrhage and in some instanceseventual fatality. On the other hand, uncontrolled clotting orcoagulation of the blood within vessels can also lead to seriouscomplications. In light of these and other complications related toblood clotting, there has naturally been a great desire to develop testswhich can be used to measure clotting tendencies and to determine thecause of any abnormalities, as well as methods and materials fortreating bleeding sites.

A number of tests have been developed to monitor or determine the causesof abnormal blood clotting tendencies. Although these known tests havein some instances proven acceptable to some extent, especially in lightof the desire to improve the human condition, there is a counting needfor even more sensitive blood clotting tests which give consistent andreproducible results. Additionally, improvements need to be made in thereagents for these prior art tests, which are commonly turbid or includeundesirable particulate matter, and which have solid, non-solubleactivating species, which fact is bone out by filtering these prior artreagents and noting substantial if not complete loss of coagulationactivating ability. Further, there is an ever-present need for newassays for accurately monitoring or determining clotting conditions forwhich there are no known accurate tests.

For example, one general coagulation test procedure which has beendeveloped is the activated partial thromboplastin time (also commonlyreferred to as the aPTT). Early on, a typical aPTT test was conducted byincubating a citrated plasma sample in contact with a solid material,such as glass, celite or kaolin, known to activate Factor XII (HagemannFactor). Then, Ca²⁺ ion and a platelet substitute (i.e. a phospholipidsuch as a cephalin derived from brain tissue or soy bean) was added tothe sample, and the time necessary for the sample to clot was measured.More recently, commercial aPTT reagents have been developed whichinclude the platelet substitute and a chemical known to activateHagemann Factor, such as ellagic-acid. For example, commercial aPTTreagents are available from Dade Division of Baxter Travenol, of MiamiFla., Ortho Diagnostics of Raritan, N.J., and Nyegaard A. S. of Norway.In a typical use, these commercial aPTT reagents are added to a citratedplasma sample which is then incubated for a period of time (commonlyabout 5 minutes) for activation to occur. Ca²⁺ is then added to thesample, often in the form of CaCl₂, and the time necessary for clottingis measured. However, these aPTT reagents have proven to beunsatisfactory in many aspects because they are not sensitive enough toheparin, Factor deficiencies, or other causes of abnormal clottingtendencies. Also, these commercial reagents give sporadic andunreproducible results in some instances, problems which some haveattributed to the presence of particulate or otherwise non-dissolvedmatter in the reagents. Also, the activator in these commercial reagentsis not in solution, a fact which is confirmed by filtering the reagentsand noting a substantial or complete loss of activating behavior.

Another generally known coagulation test procedure is the ActivatedWhole Blood Coagulation Time (AWBCT). Typical known AWBCT tests areperformed by placing a whole blood specimen in a test tube containingsolid particulate material such as celite for activation of HagemannFactor. Thereafter, the sample is heated and agitated, and the timenecessary for the sample to clot is measured. As with the known aPTTtests, however, these prior art AWBCT tests often give unreliable andunreproducible results. This could result from activation with the solidparticulate material. Activation in this manner tends to be non-uniformand to interfere with normal coagulation mechanisms due to adsorption ofFactors and other materials to the solid particles.

In addition to the above-noted shortcomings of known tests and reagents,there has been a more wholesale failure in this area in providing bloodclotting tests which are sensitive to the activity of blood platelets inthe coagulation scheme. This is despite the existence of very numerousdisorders and treatments which impact platelet activity.

For example, one condition which is known to affect platelet activity issystemic lupus. This form of lupus is thought to be attributable to thepresence of "lupus anticoagulant," which is an antiphospholipid antibodywhich inhibits the action of Platelet Factor 3 (PF3) in the coagulationmechanism. One test which has been suggested for use in detecting lupusanticoagulant is an ACT test performed on platelet poor plasma usingkaolin to activate Hagemann Factor. T. Exner et al., British Journal ofHaematology, 1978, 40, 143-51. While Exner et al. reported successfullydetecting lupus anticoagulant using their method, it nonethelessinvolves using solid particulate material for activation which, asdiscussed above, can lead to decreased sensitivity and consistency fromtest to test.

The Tissue Thromboplastin Inhibition Procedure (TTI), M. Boxer et al.,Arthritis Rheum. 19:1244 (1976); M. A. Schleider et al., Blood, 1976,48, 499-509, and the Platelet Neutralization Procedure (PNP), D. A.Triplett et al., A.J.C.P., 79, No. 6, 678-82 (June 1983), have also beensuggested for use in detecting the presence of lupus anticoagulant.However, D. A. Triplett et al. demonstrated that the TTI procedure isnot specific for lupus anticoagulauts and thus does not provide adesirable test for detecting systemic lupus. Additionally, the PNP,while having been demonstrated to be sufficiently sensitive forqualitative determination of lupus anticoagulant, see V. Dayton et al.,Laboratory Medicine, January 1990, pp. 30-32, does not provide a testfor qualitative and quantitative study of platelet activity, and reliesupon the addition of freeze-thawed platelets to neutralize the lupusanticoagulant.

In addition to detection of systemic lupus, there are also many otherconditions which contribute to the need and desire for sensitive,reliable tests for platelet activity. For instance, it has long beenknown that aspirin (ASA) imhibits the activity of platelets in thecoagulation system by suppressing their release of PF3. This, in turn,can lead to extended coagulation times for blood and plasma of patientstaking aspirin. Nonetheless, as is well known, aspirin has been widelyused as a pain killer and anti-inflammatory drug. Additionally, therehas been a recent trend in medicine to prescribe a daily regimen ofaspirin to reduce risk of heart attack In fact, it has been estimatedthat over 20 million people in the U.S. presently take at least oneaspirin a day for this reason. Further, recent suggestions have beenmade that sufferers of migraine headaches can benefit from a dailyregimin of aspirin, and this could lead to over two million additionalpersons in the U.S. on daily aspirin therapy. This extensive and rapidlygrowing use of aspirin, which has heretofore somewhat recklesslyproceeded without monitoring its effect on the patients' platelets,gives rise to an urgent need for sensitive tests which can be used tomonitor aspirin therapy.

Another driving force for the development of good tests for plateletactivity is the existence of platelet function abnormalities inpatients. As an example, it has been discovered that full-term pregnancypre-eclamptic women often have prolonged bleeding times. This has beenattributed to low platelet counts, and also in some instances is thoughtto be due to abnormalities in platelets. See, J. Ramanathan et al.,Anesthesiol., 1989, 71, 188-91.

Additionally, in the area of quality control, a recent article points upthe need for a sensitive test which can be used to differentiateplatelet concentrates which retain functional integrity after storagefrom those which do not, and explains that there is presently noavailable method for accomplishing this. T. Hervig et al., Clin. Chem.,1990, 36, No. 1, pp. 28-31.

Moreover, the presence of antiphospholipid antibodies has beenassociated with the occurrence of premature fetal death syndrome, see,for instance, D. A. Triplett, College of American Pathologists Today,July 1989, Vol. 3, No. 7 p. 61, thus giving rise to an additional groupwhich would benefit from more sensitive and accurate tests for thepresence of antiphospholipid antibodies. Further, to date there are notests known to applicant for the quantitative determination of PlateletFactor 4 (PF4). However, as is known, PF4 neutralizes heparin, and thusan accurate determination of a patient's PF4 level should be animportant and routine part of the application and monitoring of heparintherapy.

Another area to which this invention relates is hemostatic agents, whichare are commonly used to control bleeding from wounds or from vesselsduring surgery. As an example, collagen preparations have been used astopical hemostatic agents. R. G. Mason et al., Haemostasis, 3, 31-45(1974). Mason et al. report that the collagen preparations apparentlyaccelerate formation of fibrin primarily by alteration of plateletsrather than by direct action on the soluble components of the intrinsiccoagulation system In this regard, product literature for AVITENE, amicrofibrillar collagen hemostat (MCH) available from AlconLaboratories, Inc. of Fort Worth, Tex., also states that contacting theMCH with a bleeding surface attracts platelets which adhere to thefibrils and undergo the release phenomenon to trigger aggregation ofplatelets into thrombi in the interstices of the fibrous mass.Physician's Desk Reference (1987) pp. 588-589. In addition to topicalhemostats, it is also known to use hemostatic pastes to stem bleedingfrom arteries during major surgery such as heart surgery. Despite theseknown hemostatic agents, there still exists a continuing need and desirefor improved hemostatic agents. The applicant's invention alsocontemplates an improved hemostatic agent to address thin need.

As is evident from the foregoing, there exists a continuing need fornew, as well as more sensitive, accurate and reliable coagulation assayswhich can be used to determine coagulative properties of blood orplasma. There also exist needs for improvements in reagents for clottingtests, and in materials and methods for treating bleeding. Theapplicant's inventions address these needs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a platelet reserve determination for a normal patientbefore and after aspirin therapy.

FIG. 2 shows a platelet reserve determination for a patient on coumadintherapy.

FIG. 3 shows a platelet reserve determination for a patient withdefective Platelet Factor 3 release.

FIG. 4 shows a platelet reserve determination on a platelet concentrate.

FIG. 5 shows a clotting time determination of plasma samples withvarious platelet to heparin ratios.

SUMMARY OF THE INVENTIONS

A first preferred embodiment relates to a reagent for uniform activationof intrinsic coagulation which consists essentially of an aqueoussolution of effective amounts of ellagic acid, phenol, and a suitablemetal ion. The ellagic acid and metal ion are fully solubilized, andconsistent activated plasma clotting times of less than about 40 secondscan be achieved for normal platelet-rich plasma.

A second preferred embodiment relates to a method for preparing acoagulation test reagent. This method comprises the steps of (a)preparing an aqueous solution by dissolving ellagic acid in water in thepresence of a suitable base, (b) adding phenol to said aqueous solution,and (c) after said adding phenol, adding a suitable metal ion to saidsolution, said phenol being added in amounts sufficient to inhibitformation of insoluble metal hydroxides of said suitable metal ion,whereby the overall reagent is a solution.

A third preferred embodiment relates to the applicant's discovery thattannin or propyl gallate, in combination with a suitable metal ion, forman excellent activator of intrinsic coagulation. Accordingly, thisembodiment involves a reagent for activation of intrinsic coagulationwhich contains a suitable metal ion and propyl gallate or tannin.

A fourth preferred embodiment relates to an aPTT reagent which comprisesfreeze-dried platelet substitute reconstituted with an aqueous solutioncontaining (i) a suitable hydroxy-substituted aromatic compound and (ii)a suitable metal ion. This reagent gives consistent activated partialthromboplastin times of less than about 40 seconds for normal plasma,and, importantly, the overall reagent is a solution and has far superiorsensitivity than prior aPTT reagents.

A fifth preferred embodiment relates to a coagulation test reagent kit.This kit comprises (i) a vial containing freeze-dried plateletsubstitute, and (ii) an aqueous solution comprising effective amounts of(i) a suitable hydroxy-substituted aromatic compound and (ii) a suitablemetal ion. The hydroxy-substituted compound and suitable metal ion arefully solubilized. Further, the solution and platelet substitute, whencombined in controlled amounts, form an overall reagent which is also asolution and which gives consistent aPTT values of less than about 40seconds for normal plasma and has superior sensitivity.

A sixth preferred embodiment relates to an improvement in a reagent foractivation of intrinsic coagulation which includes a suitable metal ionand a suitable hydroxy-substituted aromatic compound. In accordance withthe invention, this reagent also includes dextran sulfate in effectivemounts to increase sensitivity to heparin.

A seventh preferred embodiment relates to a platelet-sensitive methodfor assaying plasma for coagulation characteristics. This methodincludes the steps of (a) incubating at least one sample of the plasmain contact with a reagent containing effective amounts of a suitablehydroxy-substituted aromatic compound and a suitable metal ion, (b)after the incubating, adding calcium to the plasma sample to initiateclotting, and (c) after the adding calcium, measuring the time necessaryfor the sample to clot, the sample being at least essentially free fromplatelet substitute during clotting. In several preferred modes, thismethod provides assays for platelet activity, systemic lupus. PF4.platelet reserve, for signifying excessive circulating low-intensitylipoproteins, and platelet concentrate integrity. These assays werediscovered following the applicant's surprising finding that reagentscontaining suitable hydroxy-substituted aromatic compounds and metalions, heretofore known to activate Hagemann Factor, also cause therelease of Platelet Factor 3 from platelets. This discovery is contraryto earlier reports in the field that ellagic acid does not alterplatelet aggregation (platelet aggregation is generally considered apreliminary step to the release of PF3), A. Girolami et al., "Failure ofEllagic Acid to affect Platelet Aggregation in normal and Factor XIIdeficient Plasma," Blut, Band 31, Seite 107-112 (1975), or may eveninhibit platelet aggregation. See J. Aznar et al., "Effect of ContactFactor (Factor XII+Factor XI) on Aggregation of Platelets," Haemostasis3, 20-30 (1974).

An eighth preferred embodiment relates to a method for stemmingbleeding, which comprises the step of (i) applying to a bleeding site aneffective amount of a hemostatic agent including a suitable metal ionand a hydroxy-substituted aromatic compound.

The objects of the inventions are many. The inventions are intended toprovide coagulation reagents and tests which are improved insensitivity, uniformity of activation, and consistency in test results,and to provide sensitive and accurate assays for platelet activity andfor conditions which affect measured platelet activity. Additionalobjects and advantages are apparent from the discussions both above andbelow.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the purposes of promoting an understanding of the principles of theinventions, reference will now be made to certain preferred embodimentsand specific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of theinventions is thereby intended, such alterations and furthermodifications, and such further applications of the principles of theinventions being contemplated as would normally occur to one skilled inthe art to which the invention relates.

Improved Ellagic Acid Activator And Method For Preparation

As stated above, a first preferred embodiment relates to a reagent foruniform activation of intrinsic coagulation. This reagent consistsessentially of an aqueous solution of ellagic acid, phenol, and suitablemetal ion. The ellagic acid and suitable metal ion are fullysolubilized, and consistent activated plasma clotting times of less thanabout 40 seconds can be achieved for normal platelet-rich plasma usingthe reagent.

As indicated, the reagent of this embodiment is a solution. Thus, theellagic acid and suitable metal ion are fully solubilized, and theactivity of the reagent, or at least a substantial percentage thereof,preferably about 85% or greater, and more preferably about 90-100%, isretained even after filtering through a 0.45 micron MILLIPORE filter.Accordingly, when herein a reagent is described as being a "solution",this means that a reagent's activity is substantially the same beforeand after filtering, such as through a 0.45 micron MILLIPORE filter. Inthis regard, it is preferred that the reagent, after such filtration.retain at least 85%, more preferably at least 90%, and most preferablylevels approaching 100% of its original unfiltered activity. Thisretention of activity is evidenced by the similar respective clottingtimes for plasma obtained using the reagent before, and afterfiltration, as is further illustrated in specific Example 34 below.

Further, and also importantly, the reagent gives consistent activatedplasma clotting times (aPCT's) of less than about 40 seconds for normalplatelet-rich plasma (the procedure for the activated plasma clottingtime is discussed below). This consistency and time is achieved byincluding, along with the ellagic acid and phenol, an effective amountof suitable metal ion. The suitable metal ions are preferably, but notnecessarily, divalent, with preferred divalent metal ions beingtransition metal ions such as Cu²⁺, Ni²⁺, and Co²⁺, and most preferablyNi²⁺. These suitable divalent metal ions are preferably provided bytheir water soluble halogen or sulfate salts, such as cupric sulfate,nickel chloride, or cobalt chloride. Additionally, a preferredmonovalent metal ion is Cu⁺, and a preferred trivalent metal ion isFe³⁺. Ca²⁺ metal ions are less preferred, because calcium's presence insignificant Amount in the reagent, and thus during incubation of thesample, tends to prematurely initiate clotting mechanisms, thusinterfering with obtaining accurate clotting times.

As to the amounts of the ellagic acid and the suitable metal ion, anyamounts by which substantial activation of the intrinsic coagulationsystem can be achieved are acceptable. Preferably, the ellagic acid isincluded in molar concentrations of about 10⁻⁴ to 10⁻⁹, and morepreferably about 10⁻⁴ to 10⁻⁵ M. Additionally, it is preferred that thesuitable metal ion be included in about an equal molar concentration tothe ellagic acid, although concentrations greater than equal molarconcentrations are also acceptable. Further, the more preferredactivators of this embodiment include the phenol in an amount of about0.02M.

A suitable buffer to maintain physiological pH is also preferablyincluded. This buffer is preferably TRIS, although other suitablebuffers, such as HEPES, can be used.

The excellent sensitivity and activating ability of these inventiveellagic acid-based activators is born out by the excellent resultsobtained for clotting tests reported further herein using these ellagicacid-based activators alone as the reagent, as well as by the superioraPTT reagents which are formed using these activators. Specific Examples1-3 below further detail the preparation of intrinsic coagulationactivators according to this embodiment.

A second preferred embodiment is a method for preparing a coagulationtest reagent. The method comprises the steps of (a) preparing an aqueoussolution by dissolving ellagic acid in water in the presence of asuitable base, (b) adding phenol to said aqueous solution, and (c) aftersaid adding phenol, adding a suitable metal ion to said solution, saidphenol being added in amounts sufficient to inhibit formation ofinsoluble metal hydroxides of said metal ion, whereby the overallreagent is a solution. Many suitable bases which aid in the aqueousdissolution of ellagic acid are known, and accordingly could be usedwithin the scope of this invention. However, the applicant's preferredbase for this purpose is tetramethylammonium hydroxide. It is importantthat the ingredients be added in the order provided by this method, asany other order leads to inferior magenta containing insoluble materialswhich interfere with uniformity of activation and consistency of result.Additional aspects of this embodiment. including those relating to thepreferred amounts of ellagic acid and amounts and types of metal ions,are analogous to those detailed in the first embodiment discussed aboveand can also be found in Examples 1-3 below.

Propyl Gallate and Tannin Coagulation Activators

As stated above, a third preferred embodiment relates to the applicant'sdiscovery that tannin and propyl gallate, in combination with a suitablemetal ion, each provide a uniform, consistent activator of intrinsiccoagulation. Accordingly, this third embodiment relates to a reagent foractivating intrinsic coagulation; which reagent contains a suitablemetal ion and propyl gallate or tannin.

Although reagents in accordance with this embodiment need not besolutions to be effective, it is preferred that they be solutions inorder to achieve the same significant advantages discussed in connectionwith the embodiments set forth above. Propyl gallate and tannin provideadvantage over ellagic acid and similar suitable hydroxy-substitutedaromatic compounds, because they more readily dissolve and remain sodissolved in aqueous solutions.

The reagent of this embodiment also contains a suitable metal ion. Themetal ions need not be, but preferably are, divalent metal ions, withpreferred suitable divalent metal ions being transition metal ions suchas Ni²⁺, Cu²⁺ and Co²⁺, most preferably Ni²⁺. As in the firstembodiment, a preferred monovalent metal ion is Cu⁺, and a preferredtrivalent ion is Fe³⁺. The tannin and propyl gallate, and the metal ion,can be included in the reagent in any amounts by which the reagentactivates coagulation. Preferably, however, propyl gallate is includedin the reagent in molar concentrations of about 10⁻⁹ to 10⁻², and morepreferably 10⁻² to 10⁻³ M, and tannin is preferably included in thereagent in amounts of about 10⁻⁵ to about 10⁻¹ weight %, and morepreferably about 0.005 weight %. The metal ions are preferably includedin mounts of about 10⁻⁹ to 10⁻⁴ M, and more preferably about 10⁻⁴ to10⁻⁵ M. Additionally, the preferred divalent metal ions are preferablyprovided by their water soluble halogen or sulfate salts, such as cupricsulfate, nickel chloride, or cobalt chloride.

In addition to the above ingredients, the reagent of this embodimentalso preferably includes a suitable buffer to maintain physiological pH.Many acceptable buffers of this type are known, including for instance4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (also known as HEPES)and Tris(hydroxymethyl)aminomethane (also known as TRIS). Theapplicant's preferred buffer, however, is TRIS, which is most preferablyincluded in a concentration of about 0.01M.

Also, in one preferred mode, the reagent of this embodiment includesplatelet substitute, and accordingly is an activated partialthromboplastin time reagent. The preferred platelet substitute isASOLECTIN, which is a soy bean-derived phospholipid available fromAssociate Concentrates, Inc. of Woodside N.Y., although many othersuitable platelet substitutes are available, including for instancecephalin derived from rabbit or other animal brain. Additional detailsregarding the preparation of reagents of this embodiment are provided inExamples 7-9, 13-15, 19-21 and 28-30 below.

Highly Sensitive aPTT Reagents and Kits

As discussed above, prior aPTT reagents have been criticized for theirlack of sensitivity. The applicant has developed aPTT reagents havingfar superior sensitivity than prior reagents, and accordingly a fourthpreferred embodiment provides a reagent for activation of intrinsiccoagulation which comprises freeze-dried platelet substitutereconstituted with an aqueous solution containing effective activatingamounts of (i) a suitable hydroxy-substituted aromatic compound, and(ii) a suitable metal ion. This overall inventive reagent is a solution,and gives consistent aPTT values of less than about 40 seconds fornormal platelet poor plasma. Moreover, this reagent is highly sensitive,which is demonstrated by the reagent giving aPTT values of at leastabout 200 seconds for 1 u/ml heparinized normal platelet poor plasma. Itis preferred that the aqueous solution be an activator as described inthe first and third embodiments above. Further, the solution form of theoverall reagent can be demonstrated by the similar aPTT values obtainedbefore and after filtration, as is illustrated in Example 35 below.

To exemplify reagents according to this embodiment, aPTT tests wereperformed on normal human plasma using aPTT reagent which was preparedby reconstituting 0.001 g freeze-dried platelet substitute (ASOLECTIN)with 5 ml of the intrinsic coagulation activators ("IntrinsicActivators") from the Examples indicated in Table 1 below. The aPTTtests were run in the usual manner by adding 0.1 ml of the prepared aPTTreagent to a 0.1 ml sample of citrated platelet-poor normal plasma. Theplasma was then incubated at 37° C. for 5 minutes, whereafter calciumchloride was added to the sample initiate clotting, and the timenecessary for the samples to clot was measured. The results of thesetests are set forth in Table 1.

                  TABLE 1                                                         ______________________________________                                        Intrinsic Activator                                                                              aPTT (sec.)                                                ______________________________________                                        Copper/ellagic acid (Ex. 1)                                                                      24.4                                                       Nickel/ellagic acid (Ex. 2)                                                                      27.5                                                       Cobalt/ellagic acid (Ex. 3)                                                                      20.3                                                       Copper/propyl gallate (Ex. 7)                                                                    29.0                                                       Nickel/propyl gallate (Ex. 8)                                                                    29.4                                                       Cobalt/propyl gallate (Ex. 9)                                                                    30.3                                                       Copper/tannin (Ex. 13)                                                                           30.7                                                       Nickel/tannin (Ex. 14)                                                                           29.1                                                       Cobalt/tannin (Ex. 15)                                                                           28.0                                                       ______________________________________                                    

In another set of experiments, the sensitivity of the inventive aPTTreagents was compared to that of known commercial reagents. Forinstance, heparin sensitivity of a preferred aPTT reagent according tothe invention and various commercial aPTT reagents was compared.Accordingly, 0.001 g of lyophilized ASOLECTIN platelet substitute ("PS")was reconstituted with 5 ml of the applicant's preferred IntrinsicActivator prepared in Example 2 (this aPTT reagent is indicated by"PS/5ml" in Table 2). Also, 0.001 g of lyophilized ASOLECTIN wasreconstituted with only 2 ml of this Intrinsic Activator (indicated as"PS/2ml" in Table 2). These inventive aPTT reagents, and the commercialaPTT reagents identified in Table 2 below, were used to perform aPTTtests on normal human plasma and on heparinized (1 u/ml) normal humanplasma. The results demonstrate that the inventive aPTT reagents havehighly superior sensitivity to heparin, and gave aPTT values in excessof 200 seconds for 1 u/ml heparinized plasma, even ranging above 250seconds. This superior sensitivity to heparin represents a significantincrease over known aPTT reagents.

                  TABLE 2                                                         ______________________________________                                        aPTT Reagent    Normal Control                                                                           Heparinized                                        ______________________________________                                        PS/5 ml         30 sec.    345 sec.                                           PS/2 ml         28 sec.    245 sec.                                           Dade ACTIN      28 sec.    125 sec.                                           Ortho ACTIVATED 22 sec.    100 sec.                                           THROMBOFAX                                                                    ______________________________________                                    

In general, a reagent which has superior sensitivity to heparin willalso have superior sensitivity to Factor deficiencies. In another set ofexperiments, this proved to be true for the inventive reagents. Plasmasamples having Factor VIII, IX, and X deficiencies were assayed using aninventive aPTT reagent and commercial aPTT reagents. The inventive aPTTreagent was prepared by reconstituting 0.001 g of lyophilized ASOLECTINplatelet substitute "PS" with 10 ml of the Intrinsic Activator ofExample 2 (this aPTT reagent is indicated as "PS/10 ml" in Table 3). Theresults of the respective aPTT tests are given in Table 3 below. As isshown, the inventive aPTT reagents gave aPTT values in excess of 150seconds for the Factor deficient plasmas, even ranging above 200seconds. These values represent a marked improvement over known aPTTreagents.

                  TABLE 3                                                         ______________________________________                                                     aPTT for    aPTT for aPTT for                                                 Factor VIII Factor IX                                                                              Factor X                                    Reagent      deficiency  deficiency                                                                             deficiency                                  ______________________________________                                        PS/10 ml     212.5       249.0    203.2                                       Dade ACTIN   50.1        94.9     94.4                                        Ortho ACTIVATED                                                                            48.4        53.2     40.7                                        THROMBOFAX                                                                    ______________________________________                                    

A fifth preferred embodiment relates to a coagulation test reagent kitfrom which the preferred coagulation activators of the fourth embodimentabove can be conveniently and effectively prepared. This kit comprises(i) a vial containing freeze-dried platelet substitute, and (ii) anaqueous solution comprising effective amounts of (i) a suitablehydroxy-substituted aromatic compound and (ii) a suitable metal ion. Thehydroxy-substituted aromatic compound and the metal ion are fullysolubilized. Further, the aqueous solution and platelet substitute, whencombined in controlled amounts, form a reagent which is also a solutionand which gives consistent aPTT values of less than about 40 seconds fornormal plasma, and which gives aPTT values of at least about 200 secondsfor 1 u/ml heparinized normal platelet poor plasma. Preferably, theplatelet substitute is included in the vial in an amount wherebyreconstitution with about 2 ml to about 10 ml of the solution forms areagent giving the indicated aPTT values. Further preferred aspects ofthis embodiment correspond to those of the fourth embodiment discussedabove.

The applicant has also discovered that the preferred aPTT reagents andkits according to the fourth and fifth embodiments above can be used insensitive assays to detect lupus anticoagulant. Thus, these reagents andkits form the basis for a method for detecting the presence of lupusanticoagulant in subject plasma, which comprises the steps of (a)incubating platelet poor subject plasma in contact with a reagentaccording to the fourth embodiment above, (b) adding calcium to theplatelet poor subject plasma in effective amounts to initiate clotting,and (c) measuring the time necessary for the platelet poor subjectplasma to clot. Typically, aPTT times at least about 5 seconds greaterthan those obtained for normal controls can signify the presence oflupus anticoagulant. In the applicant's work, using this inventivemethod, lupus anticoagulant plasmas have typically exhibited aPTT timesin excess of 50 seconds, and often in excess of 100 seconds or more.

Heparin Sensitive Reagents with Dextran Sulfate

As stated above, a sixth preferred embodiment relates to an improvementin a reagent for activation of intrinsic coagulation. In accordance withthis embodiment, a coagulation activating reagent containing a suitablemetal ion and a suitable hydroxy-substituted aromatic compound, alsocontains dextran sulfate in effective mounts to increase sensitivity ofthe reagent to heparin.

Although not required, it is preferred that the reagents according tothis embodiment be solutions, as this term is defined above. Further,although other suitable hydroxy-substituted compounds can be used,reagents according to this embodiment preferably contain ellagic acid,propyl gallate or tannin. The respective preferred amounts of thesethree compounds in the reagents of this embodiment correspond to theirpreferred amounts in the reagents of embodiments described above.Similarly, the suitable metal ions are preferably divalent, withpreferred suitable divalent metal ions being transition metal ions suchas Ni²⁺, Cu²⁺ and Co²⁺, preferably included in the same amounts as inthe embodiments discussed above.

Additionally, the reagents of this embodiment, in one preferred mode,also contain platelet substitute, and thus form heparin sensitive aPTTreagents. Again, the preferred platelet substitute is ASOLECTIN, and itis preferred that the overall reagent including the platelet substitutebe a solution.

The dextran sulfate is preferably present in an amount between about0.008 and 0.012 weight mg %, more preferably about 0.01 weight mg %,although any amount which increases sensitivity to heparin issufficient. It is believed that dextran sulfate neutralizes availablePF4, thus leading to increased heparin sensitivity. Thus, othersubstances, such as protamine sulfate, which are equivalents to dextransulfate and have substantially the same properties, are contemplated asbeing within the scope of the invention.

To verify significant increase in heparin sensitivity, heparinizedplasma samples (1 u/ml) were tested for aPTT and aPCT using reagentswith ("w/") and without ("w/o") dextran sulfate ("DS"). The results areset forth in Table 4 below. The composition and preparation of thepreferred reagents used is given in the specific Examples which areindicated in parentheses in Table 4. The "aPTT w/o DS" reagent indicatedin Table 4 was prepared indentically to the reagent of Example 26,except without the addition of the dextran sulfate. The resultsdemonstrate that the addition of dextran sulfate substantially increasessensitivity to heparin.

                  TABLE 4                                                         ______________________________________                                               aPCT w/DS aPCT w/o DS                                                                              aPTT w/DS                                         Plasma (Ex. 5)   (Ex. 2)    (Ex. 26)                                                                              aPTT w/o DS                               ______________________________________                                        Heparin-                                                                             194.8     137.9      129.6   87.5                                      ized                                                                          Normal 29.4      29.0       27.7    27.7                                      Control                                                                       ______________________________________                                    

Platelet-Sensitive Assays

A seventh preferred embodiment relates to a platelet-sensitive methodfor assaying plasma. This method includes the steps of (a) incubatingthe plasma in contact with a reagent containing effective amounts of asuitable hydroxy-substituted aromatic compound and a suitable metal ionto cause the platelets to release Platelet Factor 3, (b) after theincubating, adding calcium to the plasma to initiate clotting, and (c)after the adding calcium, measuring the time necessary for the plasma toclot, the plasma being essentially free from platelet substitute duringclotting.

As the applicant has discovered, suitable hydroxy-substituted aromaticcompounds and metal ions, which were heretofore known to activateHagemann factor, also cause platelets to release Platelet Factor 3.Accordingly, the method of this embodiment provides assays which arehighly sensitive to platelets.

It is preferred that the reagent used in the incubating step be asolution as defined above. Further, the hydroxy-substituted aromaticcompound included in the reagent can be any suitable one as known in theart. Preferred, however, are ellagic add, propyl gallate and tannin, orsuitable derivatives thereof, with ellagic acid being most preferred.These compounds are preferably included in the reagent in amountscorresponding to those disclosed above for other embodiments. Likewise,the metal ion, which is preferably a divalent transition metal ion(preferred again are Ni²⁺, Cu²⁺ and Co²⁺, most preferably Ni²⁺), isincluded in the same concentrations as discussed above for otherembodiments. Of course, in order for the assay to be sensitive toplatelets, the plasma being assayed must be at least essentially freefrom, and preferably free from platelet substitute during dotting.Accordingly, the preferred reagents used in this embodiment are at leastessentially free from, and more preferably free from plateletsubstitute. As indicated above, in several preferred modes, thisplatelet-sensitive method provides assays for platelet activity,systemic lupus, PF4, platelet reserve, which can signify excessivecirculating low-density lipoproteins, and for platelet integrity. Eachof these is disused below and each provides an important assay to meetneeds in this field which prior tests have not.

I. Activated Plasma Clotting Time (aPCT) Test

In this test, the platelet-sensitive method discussed above is performedon platelet-rich plasma. Accordingly, this test provides a sensitive andaccurate measure of platelet activity of plasma. This test has beentermed the "activated plasma clotting time" (aPCT) test by applicant.

APCT tests were performed on normal platelet-rich human plasma usingpreferred Intrinsic Activators of the invention as the aPCT reagents.Accordingly, a 0.1 ml amount of the Intrinsic Activator of the Exampleindicated in Table 5 below was added to a 0.1 ml sample of normalcitrated platelet-rich plasma. The plasma was then incubated for 5minutes, whereafter calcium chloride was added to the plasma to initiateclotting. The time necessary for the samples to clot was measured, andthe results are given in Table 5 below.

                  TABLE 5                                                         ______________________________________                                        Intrinsic Activator                                                                              aPCT (sec.)                                                ______________________________________                                        Copper/ellagic acid (Ex. 1)                                                                      30.3                                                       Nickel/ellagic acid (Ex. 2)                                                                      30.0                                                       Cobalt/ellagic acid (Ex. 3)                                                                      26.5                                                       Copper/propyl gallate (Ex. 7)                                                                    30.4                                                       Nickel/propyl gallate (Ex. 8)                                                                    31.0                                                       Cobalt/propyl gallate (Ex. 9)                                                                    29.4                                                       Copper/tannin (Ex. 13)                                                                           32.4                                                       Nickel/tannin (Ex. 14)                                                                           30.9                                                       Cobalt/tannin (Ex. 15)                                                                           32.9                                                       ______________________________________                                    

II. Determination of Platelet Reserve

In a normal subject, there is an excess of platelets beyond the quantityand quality required to support normal coagulation, which is hereinreferred to as the "platelet reserve". The applicant has discovered thatthe aPCT test can be used to accurately and reliably assay for plateletreserve. Accordingly, another preferred mode of this embodiment is anassay for determining platelet reserve in subject plasma. In this assay,the activated plasma clotting times of serial dilutions of platelet richsubject plasma with platelet poor subject plasma are determined. Byanalysis of the aPCT values obtained for the dilutions, a valuerepresentative of the platelet reserve can be determined.

For example, the platelet reserve can be determined by preparingdilutions of a patient's platelet rich plasma (PRP) with the patient'splatelet poor plasma (PPP), and performing aPCT tests on the dilutions.Typically, the following dilutions can be used (PRP/PPP): 100% PRP; 50%PRP/50% PPP; 25% PRP/75% PPP; and 12.5% PRP/87.5% PPP. The aPCT timesare then plotted on a graph having % PRP on one coordinate and aPCTvalues on the other. An aPTT test is performed on PPP, and the timeobtained used as a baseline on the graph. Additionally, the aPCT valueobtained using 100% platelet-rich plasma can be used as a baseline,although using this value as a baseline may in some instances lead toinaccurate results, such as where the patient has abnormally functioningplatelets.

The applicant has found that the dilution curve will most often havelinearity extending from 12.5% to 50% PRP, and almost always from 25% to12.5% PRP. Thus, preferably a line is drawn through the aPCT values forthe 25% and 12.5% PRP dilutions. This line is extended until itintersects the baseline. The % PRP corresponding to the point ofintersection with the baseline is then subtracted from 100% to obtainthe patient's platelet reserve.

FIGS.1-3 demonstrate platelet reserve determinations performed inaccordance with this preferred embodiment. FIG. 1 shows a normal patienthaving a platelet reserve of 41% prior to aspirin therapy (line "A").After four days of aspirin therapy at 1600 milligrams/day, the patient'splatelet reserve was again determined, resulting in line "B". As can beseen, the patient's platelet reserve after the aspirin therapy decreasedto 28%. FIG. 1 thus demonstrates that the platelet reserve is anexcellent and sensitive test for monitoring aspirin therapy or othertherapies which effect the activity of platelets. The above plateletreserve figures were calculated using an aPTT baseline. Using the aPCTvalue for 100% PRP as a baseline, a value of about 44% for plateletreserve prior to aspirin therapy is obtained, which is a good estimateof platelet reserve and does not require the additional step ofperforming an aPTT test.

FIG. 2 shows a platelet reserve determination on a patient on coumadintherapy. The patient's platelet reserve was 8%, well below the normal ofabout 30% to 40%. Thus, it was demonstrated that the platelet reservetest can also be performed on patients on coumadin or like therapy.

FIG. 3 shows a platelet reserve determination on a patient withdefective Platelet Factor 3 release. As is shown, the patient's plateletreserve was determined to be 0%. Accordingly, the platelet reserve canalso be used to detect functional defects of platelets.

In another experiment, it was demonstrated that a platelet reservedetermination could be used to assay the functional integrity ofplatelets in a platelet concentrate. Accordingly, a platelet concentratewas obtained from Universal Reagents of Indianapolis, Ind. Theconcentrate had a platelet count of 2,080,000/ml. This concentrate wasdiluted 50% with platelet poor plasma. This 50% diluted concentrate wasthen subjected to a platelet reserve assay as was performed for FIGS.1-3. The results are shown in FIG. 4, and demonstrate that the 50%dilution had a platelet reserve of 39%. Accordingly, taking into accountthe 50% dilution factor, the platelet concentrate was determined to havea platelet reserve of 78% (39%×2). It will be understood thatalternative analyses can be used to determine platelet reserve from therespective aPCT values obtained for the dilutions, and that graphicalrepresentation is unnecessary.

III. Detection of Lupus Anticoagulant

It has been discovered that the platelet-sensitive method provides asensitive and specific assay for lupus anticoagulant when performed onplatelet-poor plasma. For example, Table 6 summarizes studies whichdemonstrate this specificity and sensitivity. Table 6 shows the clottingtimes obtained for various abnormal plasmas, including lupus plasmas(platelet poor), using the intrinsic activator of Example 2 (Ni/ellagicacid) as the reagent, as compared to aPTT values obtained for these sameplasmas (using an aPTT reagent prepared as in Example 26 except withoutthe addition of dextran sulfate). The variety of plasma's were obtainedfrom Universal Reagents of Indianapolis, Ind. As is demonstrated, thelupus test of this preferred mode has outstanding specificity andsensitivity for and to lupus anticoagulant.

                  TABLE 6                                                         ______________________________________                                        Type of Plasma     aPTT   aPCT                                                ______________________________________                                        platelet rich plasma                                                                             25.8   26.0                                                platelet poor plasma                                                                             26.2   71.2                                                Lupus anticoagulant                                                                              39.0   263.1                                               Lupus anticoagulant                                                                              48.7   269.5                                               ANA speckled       39.7   67.4                                                ANA speckled       32.2   62.9                                                ANA homogenous     33.8   65.3                                                ANA homogenous     32.2   54.6                                                nDNA Antibody      33.9   75.7                                                SCL-70             34.3   46.8                                                RNP                44.7   71.2                                                SSA Antibody       19.9   37.0                                                SSB Antibody       37.7   61.5                                                ______________________________________                                    

Table 7 below compares the lupus anticoagulant sensitivity of theapplicant's preferred lupus test (using the Intrinsic Activator ofExample 2 as the reagent), to the sensitivity obtained using variouscommercial aPTT reagents. The plasmas tested were all platelet poor. Asshown, the preferred lupus test of this mode is far superior thancommercial aPTT reagents and tests in the detection of lupusanticoagulant.

                  TABLE 7                                                         ______________________________________                                                              Ortho    Dade                                           Type of Plasma Ex. 2  aPTT     ACTIN Biodata                                  ______________________________________                                        Lupus anticoagulant                                                                          174.4  31.6     44.7  51.1                                     Lupus anticoagulant                                                                          221.9  32.6     46.1  61.0                                     Norm. Human Plasma                                                                           64.8   25.3     27.9  32.8                                     (platelet poor)                                                               ______________________________________                                    

IV. Assay For Lipoprotein Defect

It has been demonstrated that the applicant's preferredplatelet-sensitive method can be used to signify possible Type IIlipoprotein defects. Subjects having this condition have excessivelyhigh circulating low-density lipoproteins. In this assay, theplatelet-sensitive method is performed on platelet-poor subject plasmaand on platelet-rich subject plasma (this latter test is simply an aPCTtest). The two measured clotting times are then compared, whereby thelack of a significant difference can signify excessive circulatinglow-density lipoprotein.

For example, such an assay was performed on a patient suspected ofhaving a Type II lipoprotein defect. The patient's platelet rich plasmaexhibited an aPCT in accordance with the invention of 31.2 seconds(using the intrinsic activator of Example 2 as the aPCT reagent). Thepatient's platelet poor plasma demonstrated a clotting time of 33.1seconds using the same reagent, as opposed to normal values of about 60seconds or more for normal platelet poor plasma. A possible Type IIlipoprotein defect was thus signified, which was confirmed bylipoprotein electrophoresis of the patient's plasma.

V. Assay For Platelet Factor 4

The platelet-sensitive method can also be used to test for PlateletFactor 4 release in plasma. In the PF4 assay, the platelet-sensitivemethod is performed on plasmas having varying ratios of platelets toheparin units, whereby neutralization of the lieperin present byPlatelet Factor 4 released by the platelets can be observed. Of course,the varying ratios can be obtained by adjusting the level of platelets,or the level of heparin, or by adjusting the level of both.

For example, in one method, platelet rich subject plasma and at leastone dilution of platelet rich subject plasma (with platelet poor plasma)are equally heparinized. The measured clotting times are then compared,whereby a significantly lower clotting time for the heparinized plateletrich plasma as compared to the dilution can indicate release ofsufficient Platelet Factor 4 in the platelet rich plasma to neutralizethe heparin. In an experiment using this approach, two plasma samples,each containing 1 unit of heparin per ml, were prepared. The firstsample was 100% platelet rich plasma, and the second was 50% plateletrich plasma and 50% platelet poor plasma. The clotting times obtainedusing the intrinsic activator of Example 2 as a reagent were 31.3seconds for the 100% PRP, and 208.6 seconds for the 50% dilution. Therelatively short clotting time for the 100% PRP indicates the release ofsufficient PF4 to neutralize the heparin. The relatively long clottingtime for the 50% dilution indicates that less PF4 has been released (dueto the reduced concentration of platelets) so that less heparin wasneutralized, leading to the longer clotting time. The ability of theplasma used in the two samples to release PF4 was thus demonstrated. Hadthe plasma had a defect in PF4 release or function, the aPCT value forthe 100% PRP would have been significantly longer.

It will be appreciated that significantly more precise measures of thelevels of PF4 in plasma samples can be obtained by assaying plasmashaving several ratios of platelets to heparin to determine moreprecisely the point of neutralization of the heparin by PF4.Accordingly, in another experiment. dilutions were prepared havingeffectively the levels of heparin per ml of PRP indicated in Table 8below. The clotting times were obtained for each of these dilutionsusing the intrinsic activator of Example 2 as the reagent. The resultsare tabulated in Table 8, and graphically represented in FIG. 5. Theseresults demonstrate the sufficient release of PF4 in the patient'splatelet-rich plasma to neutralize between 3 and 4 units of heparin perml. Thus the inventive PF4 assay provides a way to accurately determinePF4 levels in plasma samples.

                  TABLE 8                                                         ______________________________________                                        U/Heparin/ml  Clotting Time (sec)                                             ______________________________________                                        0             30.1                                                            0.5           30.1                                                            1.0           29.6                                                            1.5           28.8                                                            2.0           27.3                                                            3.0           39.4                                                            4.0           186.8                                                           ______________________________________                                    

Activated Whole Blood Clotting Time

The applicant has also demonstrated that the preferred intrinsicactivators can be used in performing activated whole blood clotting timetests (A.W.B.C.T). Accordingly, the activated whole blood clotting timeof fresh human blood was determined by incubating 0.1 ml fresh humanblood and 0.1 ml of the intrinsic activator of Example 2 in a Fibrometerfor 5 minutes. 0.1 ml 0.02M CaCl₂ was added, whereafter the A.W.B.C.T.value obtained was 43.4 seconds. Similar tests on two additional freshwhole blood samples gave A.W.B.C.T. values of 38.4 and 38.9 seconds.Thus the inventive Intrinsic Activators are useful for whole blood aswell as plasma-based tests.

Method and Materials to Stem Bleeding

In yet another set of experiments, the applicant has discovered thatbleeding from wounds can be stopped up to six time faster by applying tothe bleeding site an effective mount of a hemostatic agent including asuitable metal ion and a hydroxy-substituted aromatic compound.Preferred are reagents according to the first embodiment above, whichcan in this embodiment also optionally include a platelet substitute,preferably ASOLECTIN, and also preferred are reagents according to thethird embodiment above.

For example, in one experiment the intrinsic activator of Example 2 wasapplied to a portion of fibrous material (e.g. filter paper) and thismaterial was then applied to a bleeding finger wound. The wound stoppedbleeding in about 16 seconds. Another bleeding finger wound treatedsimilarly with an unmodified portion of the fibrous material stoppedbleeding only after 94 seconds. It was thus demonstrated that the methodof this embodiment provides an outstanding way to stem bleeding.

It will be understood that there are other materials to which thereagents can be applied in particular bleeding applications. Forinstance, it is known to prepare pastes by reconstitutingcryoprecipitates of human blood with thromboplastin for application tobleeding sites during surgery such as coronary surgery. In one preferredmode of this embodiment, the indicated reagents are used in thereconstitution of the cryoprecipitates in the preparation of suchpastes. The resulting pastes have superior ability to slow or stopbleeding, and thus these materials themselves constitute a furtherinventive embodiment.

The following Examples are given in order to further illustrate theinvention.

EXAMPLE 1 Ellagic Acid and Copper II Intrinsic Activator

0.18 g tetramethylammonium hydroxide were dissolved in 1,000 ml water.0.034 g ellagic acid were then dissolved in this tetramethylammoniumhydroxide solution. 1.5 g phenol were then dissolved in thetetramethylammonium hydroxide-ellagic acid solution, whereafter 1.0 ml0.1M cupric sulfate were added and the resulting solution mixed for 10minutes with teflon coated stirring bar on a stir-plate. 1.2 g TRISbuffer were then dissolved into the solution. The resulting solution wasclear and free from any visible particulate or other suspended matter.

EXAMPLE 2 Ellagic Acid and Nickel Intrinsic Activator

Example 1 was repeated except 1.0 ml 0.1M nickel chloride was usedinstead of the 1.0 ml 0.1M cupric sulfate to form an ellagic add/nickelcoagulation activator which was also a clear solution free from visibleparticulate or other suspended matter.

EXAMPLE 3 Ellagic Acid and Cobalt Intrinsic Activator

Example 1 was repeated except 1.0 ml 0.1M cobalt chloride was usedinstead of the 1.0 ml 0.1M cupric sulfate, and the TRIS buffer was notadded. A clear ellagic acid/cobalt coagulation activator Solution wasformed. This unbuffered activator performs similarly to the bufferedactivators in Examples 1 and 2. The buffer further stabilizes theactivators over time.

EXAMPLE 4 Heparin Sensitive Intrinsic Activator with Ellagic Acid,Dextran Sulfate, and Copper II

Example 1 was repeated except 1.0 ml 100 mg/dl dextran sulfate was addedalong with the cupric sulfate to form a coagulation activator sensitiveto heparin,

EXAMPLE 5 Heparin Sensitive Intrinsic Activator with Ellagic Acid,Dextran Sulfate, and Nickel

Example 2 was repeated except 1.0 ml 100 mg/dl dextran sulfate was addedalong with the nickel chloride. A heparin sensitive coagulationactivator with ellagic acid, dextran sulfate, and nickel was thusformed.

EXAMPLE 6 Heparin Sensitive Intrinsic Activator with Ellagic Acid,Dextran Sulfate and Cobalt

Example 3 was repeated except 1.0 ml 100 mg/dl dextran sulfate was addedalong with the cobalt chloride to form a heparin sensitive coagulationactivator with ellagic acid and cobalt.

EXAMPLE 7 Propyl Gallate and Copper II Intrinsic Activator

0.5 g propyl gallate were dissolved in 1,000 ml water. 1.0 ml 0.1Mcupric sulfate were then added, whereafter the solution was mixed for 10minutes with teflon-coated stir bar. 1.2 g TRIS buffer were thendissolved in this solution, which was thereafter stirred for anadditional 10 minutes. A propyl gallate/copper coagulation activator wasformed as a clear solution without any visible suspended materials.

EXAMPLE 8 Propyl Gallate and Nickel Intrinsic Activator

Example 7 was repeated except 1.0 ml 0.1M nickel chloride was usedinstead of the cupric sulfate. A clear solution-form coagulationactivator with propyl gallate and nickel was formed.

EXAMPLE 9 Propyl Gallate and Cobalt Intrinsic Activator

Example 7 was repeated except 1.0 ml 0.1M cobalt chloride was used theplace of the cupric sulfate to form a propyl gallate/cobaltsolution-form coagulation activator.

EXAMPLE 10 Heparin Sensitive Intrinsic Activator with Propyl Gallate,Dextran Sulfate and Copper II

Example 7 was repeated except 1.0 ml 100 mg/dl dextran sulfate was addedalong with the cupric sulfate. A heparin sensitive coagulation activatorwith propyl gallate, dextran sulfate and copper was thus formed.

EXAMPLE 11 Heparin Sensitive Intrinsic Activator with Propyl Gallate,Dextran Sulfate and Nickel

Example 8 was repeated except 1.0 ml 100 mg/dl dextran sulfate was addedalong with the nickel chloride to form a heparin sensitive coagulationactivator containing propyl gallate, dextran sulfate and nickel.

EXAMPLE 12 Heparin Sensitive Coagulation Activator with Propyl Gallate,Dextran Sulfate and Cobalt

The procedure of Example 9 was repeated except 1.0 ml 100 mg/dl dextransulfate was added along with the cobalt chloride. A solution-formcoagulation activator was thus formed which was sensitive to heparin andcontained propyl gallate, dextran sulfate, and cobalt.

EXAMPLE 13 Tannin and Copper II Intrinsic Activator

0.5 g tannin were dissolved in 1,000 ml reagent water. 1.0 ml 0.1Mcupric sulfate were added whereafter the resulting solution was mixedfor 10 minutes with a teflon coated stirring bar on a stir-plate. Then,1.2 g TRIS buffer were added, and the resulting solution mixed for 10minutes with a teflon coated stir-bar. A clear solution coagulationactivator with tannin and copper was thus formed containing no visiblesuspended matter.

EXAMPLE 14 Tannin and Nickel Intrinsic Activator

Example 13 was repeated except 1.0 ml 0.1M Nickel chloride was used inthe place of the cupric sulfate thus forming a solution-form coagulationactivator with tannin and nickel.

EXAMPLE 15 Tannin and Cobalt Intrinsic Activator

Example 13 was repeated except 0.1 ml 0.1M cobalt chloride was usedinstead of the cupric sulfate. Also, 2,5 g HEPES hemi sodium salt wereadded instead of the TRIS, to thus make a coagulation activator insolution form containing tannin and cobalt.

EXAMPLE 16 Heparin Sensitive Intrinsic Activator with Tannin, DextranSulfate and Copper II

Example 13 was repeated, but 1.0 ml 100 mg/dl dextran sulfate was addedalong with the cupric sulfate to form a heparin sensitive coagulationactivator solution containing tannin, dextran sulfate and copper.

EXAMPLE 17 Heparin Sensitive Intrinsic Activator with Tannin, DextranSulfate and Nickel

Example 14 was repeated except 1.0 ml 100 mg/dl dextran sulfate wasadded along with the nickel chloride. A heparin sensitive coagulationactivator solution containing tannin, dextran sulfate and nickel wasformed.

EXAMPLE 18 Heparin Sensitive Intrinsic Activator with Tannin, DextranSulfate and Cobalt

Example 15 was repeated, but 1.0 ml 100 mg/dl dextran sulfate-was addedalong with the cobalt chloride to make a heparin sensitive coagulationactivator with tannin, dextran sulfate and cobalt.

EXAMPLE 19 APTT Reagent Including Propyl Gallate and Copper II Activator

In this example, a solution-form aPTT reagent was formed which includeda propyl gallate and copper coagulation activator. Accordingly, 0.5 gpropyl gallate were dissolved in 1,000 ml reagent water. 1.2 g TRISbuffer were then dissolved in this solution whereafter it was mixed for10 minutes with a teflon coated stirring bar on a stir-plate. 1.0 gAsolectin and 200 ml above solution were placed in a Waring blender andblended at full speed for 1.0 minute. The resulting emulsion wasreturned to the original solution and mixed for 10 minutes, whereafter1.0 m 0.1M cupric sulfate was added and the resulting solution was mixedfor an additional 10 minutes. A solution-form a reagent was formed whichwas clear and free from the cloudiness or suspended material commonlyobserved in commercial aPTT reagents.

EXAMPLE 20 APTT Reagent Containing Propyl Gallate and Nickel Activator

Example 19 was repeated except 1.0 ml 0.1M nickel chloride was usedinstead of the cupric sulfate, thus forming a solution-form aPTT reagentcontaining a propyl gallate and nickel coagulation activator.

EXAMPLE 21 APTT Reagent Containing Propyl Gallate and Cobalt Activator

Example 19 was repeated except 1.0 ml 0.1M cobalt chloride was used inthe place of the cupric sulfate. A solution-form aPTT reagent containinga propyl gallate and cobalt activator was thus formed.

EXAMPLE 22 Heparin Sensitive APTT Reagent Containing Dextran Sulfate andA Propyl Gallate Copper II Activator

The preparation of Example 19 was repeated except 1.0 ml 100 mg/dldextran sulfate was added along with the cupric sulfate to render theaPTT reagent heparin sensitive. The reagent formed was a solutioncontaining the propyl gallate/copper activator.

EXAMPLE 23 Heparin Sensitive APTT Reagent Containing Dextran Sulfate andA Propyl Gallate/Nickel Activator

Example 20 was repeated except 1.0 ml 100 mg/dl dextran sulfate wasadded along with the nickel chloride. Accordingly, a solution-form aPTTreagent was prepared which was sensitive to heparin and which containeda propyl gallate/nickel activator.

EXAMPLE 24 Heparin Sensitive APTT Reagent Containing Dextran Sulfate andA Propyl Gallate/Cobalt Activator

The preparation of Example 21 was repeated, but 1.0 ml 100 mg/dl dextransulfate was added with the cobalt chloride. A solution-form aPTT reagentwas formed containing a propyl gallate/cobalt coagulation activator andwhich was sensitive to heparin.

EXAMPLE 25 Heparin Sensitive APTT Reagent Containing Dextran Sulfate andan Ellagic Acid/Copper II Activator

0.18 g tetramethylammonium hydroxide were dissolved in 1,000 ml reagentwater. 0.034 g ellagic acid were dissolved in the tetramethylammoniumhydroxide solution, after which 1.5 g phenol were dissolved in thetetramethylammonium hydroxide-ellagic acid solution. 1.0 g Asolectin and200 ml of above solution were then added to a Waring blender and blendedfor 1 minute at top speed. The resulting emulsion was then returned tothe original solution and mixed for 10 minutes with a teflon coatedstirring bar on a stir-plate, whereafter 1.0 ml 0.1M cupric sulfate and1.0 ml 100 mg/dl dextran sulfate were added and the resulting solutionmixed for 10 minutes more. The resulting heparin sensitive aPTT reagentcontaining an ellagic acid/copper activator was a solution without anyvisible suspended material. Another identical reagent was prepared,except 1.2 g TRIS buffer were added in the final step.

EXAMPLE 26 Heparin Sensitive APTT Reagent Containing Dextran Sulfate andan Ellagic Acid/Nickel Activator

Example 25 was repeated except 1.0 ml 0.1M nickel chloride was usedplace of the cupric sulfate. A solution-form heparin sensitive aPTTreagent an ellagic acid/nickel activator was thus prepared.

EXAMPLE 27 Heparin Sensitive APTT Reagent Containing Dextran Sulfate andan Ellagic Acid/Cobalt Activator

Example 25 was repeated except 1.0 ml 0.1M cobalt chloride was usedinstead of the cupric sulfate to prepare a solution-form heparinsensitive aPTT reagent with an ellagic acid/cobalt activator.

EXAMPLE 28 APTT Reagent Containing a Tannin/Copper II CoagulationActivator

0.5 g tannin were dissolved in 1,000 ml reagent water. 4.9 g HEPES 0.5sodium were added to the resulting solution which was then mixed for 10minutes with a teflon coated stirring bar on a stir-plate. 1.0 gAsolectin and 200 ml of the above solution were then placed in a Waringblender and blended at full speed for 1.0 minute. The resulting emulsionwas returned to the original solution and mixed for 10 minutes.Thereafter, 1.0 ml 0.1M cupric sulfate was added and the solution mixedfor another 10 minutes. Formed was an aPTT reagent in solution form freefrom visible suspended materials, and containing a tannin/coppercoagulation activator.

EXAMPLE 29 APTT Reagent Containing a Tannin/Nickel Coagulation Activator

Example 28 was repeated except 1.0 ml 0.1M nickel chloride was usedinstead of the cupric sulfate. A solution-form aPTT reagent was thusprepared having a tannin/nickel coagulation activator.

EXAMPLE 30 APTT Reagent Containing

A Tannin/Cobalt Coagulation Activator

Example 28 was repeated except 1.0 ml 0.1M cobalt chloride was usedinstead of the cupric sulfate. A solution-form aPTT reagent was thusprepared having a tannin/cobalt coagulation activator.

EXAMPLE 31 Heparin Sensitive APTT Reagent Containing Dextran Sulfate andA Tannin/Copper II Activator

Example 28 was repeated except 1.0 ml 100 mg/dl dextran sulfate wasadded along with the cupric sulfate. A heparin sensitive solution-formaPTT reagent was thus formed having a tannin/copper activator.

EXAMPLE 32 Heparin Sensitive APTT Reagent Containing Dextran Sulfate andA Tannin/Nickel Activator

Example 29 was repeated except 1.0 ml 100 mg/dl dextran sulfate wasadded with the nickel chloride to form a heparin sensitive aPTT reagentcontaining a tannin/nickel activator and being in solution form.

EXAMPLE 33 Heparin Sensitive APTT Reagent Containing Dextran Sulfate andA Tannin/Cobalt Activator

Example 30 was repeated, but with 1.0 ml 100 mg/dl dextran sulfate beingadded along with the cobalt chloride. A solution-form heparin sensitiveaPTT reagent was thus formed including a tannin/cobalt coagulationactivator.

EXAMPLE 34 Intrinsic Activators are in Solution

An amount of a coagulation activator of Example 2 was filtered through a0.45 micron MILLIPORE filter, whereupon no residue on the filter wasobserved. Using the filtered coagulation activator, an aPCT test wasperformed on normal platelet rich plasma. The aPCT value using thefiltered coagulation activator was 29.3 seconds. A similar aPCT was runusing an unfiltered amount of the same coagulation activator, and avalue of 29.4 seconds was obtained. It was thus demonstrated that theactivator was in solution form.

EXAMPLE 35 Solution Form of Inventive aPTT Reagents

0,001 g of ASOLECTIN was reconstituted with 5 ml of the coagulationactivator of Example 2. The resulting aPTT, reagent was then filteredthrough a 0.45 micrometer MILLIPORE filter whereupon no residue wasobserved. An aPTT value of 27.2 for normal platelet rich plasma wasobtained using the filtered reagent. Using a similarly prepared butnon-filtered aPTT reagent, an aPTT value of 27.5 was obtained for normalplatelet rich plasma, thus demonstrating that the total aPTT reagent issoluble.

While the inventions have been described in detail in the foregoingdescription, the same is to be considered as illustrative and notrestrictive in character, it being understood that only the preferredembodiments have been described, and that all changes and modificationsthat come within the spirit of the invention are desired to beprotected.

I claim:
 1. A method of stemming bleeding from a bleeding site,comprising the step of applying to the bleeding site an effective amountof a hemostatic agent comprising a metal ion selected from the groupconsisting of Ni⁺⁺, Co⁺⁺, Cu⁺⁺ Cu⁺ and Fe⁺⁺⁺ and a hydroxy-substitutedaromatic compound selected from the group consisting of propyl gallateand tannin.
 2. The method according to claim 1 wherein the hemostaticagent is a solution comprising the metal ion and propyl gallate ortannin.
 3. The method according to claim 1 wherein the metal ion is adivalent transition metal ion.
 4. The method according to claim 3wherein the metal ion is present in an amount in the range of from about10⁻⁴ to about 10⁻⁵ M.
 5. The method according to claim 1 wherein thehydroxy-substituted aromatic compound is tannin.
 6. The method accordingto claim 5 wherein the tannin is present in an amount in the range offrom about 10⁻⁵ to about 10⁻¹ weight %.
 7. The method according to claim1 wherein the hemostatic agent further comprises a buffer to maintainphysiological pH.
 8. The method according to claim 1 wherein thehemostatic agent further comprises a platelet substitute.
 9. The methodaccording to claim 1 wherein the hemostatic agent is applied to afibrous material which is applied to the bleeding site.
 10. The methodaccording to claim 1 wherein the hemostatic agent is a paste comprisingreconstituted cryoprecipitates of human blood and thromboplastin.
 11. Amethod for stemming bleeding from a bleeding site, comprising the stepof applying to the bleeding site an effective amount of a hemostaticagent comprising a metal ion selected from the group consisting of Ni⁺⁺,Co⁺⁺, Cu⁺⁺, Cu⁺ and Fe⁺⁺⁺, and propyl gallate.
 12. The method accordingto claim 11 wherein the propyl gallate is present in an amount in therange of from about 10⁻² to about 10⁻³ M.
 13. A hemostatic agent forstemming bleeding from a bleeding site comprising a metal ion selectedfrom the group consisting of Ni⁺⁺, Co⁺⁺, Cu⁺⁺ Cu⁺ and Fe⁺⁺⁺ and ahydroxy-substituted aromatic compound selected from the group consistingof propyl gallate and tannin.
 14. The hemostatic agent according toclaim 13 wherein the metal ion is a divalent metal ion.
 15. Thehemostatic agent according to claim 14 further comprising a fibrousmaterial to which the divalent metal ion and propyl gallate or tannin isapplied.
 16. A hemostatic agent for stemming bleeding from a bleedingsite comprising a paste which comprises reconstituted cryoprecipitatesof human blood, thromboplastin, a metal ion selected from the groupconsisting of Ni⁺⁺, Co⁺⁺, Cu⁺⁺ Cu⁺ and Fe⁺⁺⁺, and propyl gallate ortannin.